Vitro toxicology kit and method therefor

ABSTRACT

A method is described for producing tissue from cells, such as keratinocyte, epithelial or endothelial cells, in vitro. Cells are grown on a porous cell-growth substrate having specific growth factors therein. Cells can be grown to form a confluent monolayer or differentiaed tissue which can be used for studying cell toxicology.

RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 07/347,448,filed May 4, 1989, issued as U.S. Pat. No. 4,996,154 the teachings ofwhich are incorporated by reference herein.

BACKGROUND OF THE ART

A number of methods for culturing mammalian cells of different tissueorigins have been reported. However, many of these cells are difficultto grow in vitro and, when grown, are not morphologically similar to invivo tissue.

Green and Rheinwald U.S. Pat. No. 4,016,036, 1977 describe a procedurefor serially cultivating keratinocyte cells grown in the presence ofmitotically inhibited fibroblasts. Without a second cell type (e.g., 3T3fibroblasts), the keratinocyte cultures were neither uniform nordifferentiated. A major disadvantage of using tissue produced by thismethod for in vitro toxicology and other studies is the presence offibroblasts. Although the fibroblasts are mitotically inhibited, theyare still metabolically active. As such, the metabolic activity and/orcellular components of the fibroblasts interfere with assays for cellsunder study.

Woodley and co-workers (Joint Meeting of Amer. Chem. Soc. Cell Biol. andAmer. Soc. Biochem. Mol. Biol., Abstract 4536, page 798a, Jan. 29-Feb.2, 1989, San Francisco, Calif.) describe a method for growingkeratinocyte cells on collagen without the use of a second cell type. Amedium containing epidermal growth factor and bovine pituitary extractwas employed to facilitate cell growth. Epidermal tissue grown by thismethod, however, cannot be raised to the air/liquid interface sincethere is no means for providing nutrition to the tissue.

A method for growing keratinocyte cells at the air/liquid interface hasbeen described by Bernstam, I. L. et al., In Vitro Cell Dev. Biol.22:695-704 (1986). Cells grown on a collagen substrate producedconfluent monolayers; however, non-uniform areas of stratification wereobserved.

Thus, it would be desirable to produce a tissue which is morphologicallysimilar to its in vivo counterpart for in vitro toxicology and otherstudies (for example, transepidermal drug transport).

SUMMARY OF THE INVENTION

This invention pertains to methods for producing tissue, in vitro, andto a substrate for growing tissue thereon. Specifically, tissue fromcells is prepared by contacting a porous cell-growth substrate with cellculture medium comprising growth factor specific for growth of cells ofinterest, such as keratinocyte cells, epithelial cells and endothelialcells. Growth factor is then contacted with the substrate underconditions whereby it is dispersed within or onto the substrate.Preferably, the cell-growth substrate comprises a microporous membranewhich is coated with cell-growth supporting material, such as collagen.Cells are subsequently seeded onto the cell-growth substrate whereby thecells contact or are in close proximity to the growth factors to form acell culture. The seeded cell-growth substrate is maintained underconditions suitable for cell growth to thereby produce tissue. Tissueproduced by the methods of this invention can be a confluent monolayerof cells or it can be a differentiated, multilayer of cells. Variousstages of tissue growth, from the formation of a confluent monolayer andthrough the stage of stratification and terminal differentiation, can beachieved by manipulating the conditions for cell growth. The culture canbe grown submerged in culture medium or it can be raised to theair/liquid interface to mimic in vivo conditions. In either case, thecells form tissue which is evenly distributed over the substrate,uniformly stratified, and terminally differentiated. Tissue produced bythe methods of this invention is sufficiently morphologically similar toits in vivo counterpart so that it is useful for in vitro toxicology.

This invention also pertains to a method for determining toxic effectsof a substance on tissue produced by the methods of this invention andto toxicological kits comprising the tissue grown on a cell-growthsubstrate having growth factors thereon. Alteratively, the kit willcomprise the cell-growth substrate and cells of interest for growing onthe substrate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a transmission electron micrograph (TEM) which shows theair/liquid interface of a keratinocyte sheet with cornified envelopesgrown according to the methods of this invention.

FIG. 2 is a TEM which shows the basal portion of the keratinocyte sheetshown in FIG. 1.

FIG. 3 shows a hematoxylin and eosin stained histological cross-sectionof a uniform sheet of keratinocytes grown on a modified, crosslinkedcollagen-coated cell-growth substrate made according to this invention.

FIG. 4 shows a dose-response curve for keratinocyte cells grown on acollagen-coated microporous cell-growth substrate according to themethods of the invention and exposed to various concentrations of sodiumdodecyl sulfate (SDS).

DETAILED DESCRIPTION OF THE INVENTION

Cells which are suitable for producing tissue by the methods of thisinvention, include but are not limited to, keratinocyte cells,hepatocyte cells, nerve cells, endothelial cells, and epithelial cellsof any tissue origin such as, corneal epithelial pulmonary epithelialcells. The cells can be from any natural tissue source (mammalian orother cell source), or they can be genetically engineered. The term"tissue" is herein intended to mean an organization of cells grown toform a confluent uniform monolayer or multilayer tissue that can bestratified and terminally differentiated.

The cell-growth substrate is critical to the proliferation anddifferentiation of cells to form tissue similar to in vivo tissue. Aporous cell-growth substrate is defined herein to be any substrate whichwill support cell growth. Suitable cell-growth substrates will have aporous structure which can facilitate diffusion of nutrients to thecells, particularly to the basal cells of the tissue. For example, theporous cell-growth substrate can contact medium and supply tissue raisedto the air/liquid interface with sufficient nutrients. The cell-growthsubstrate can be any porous natural or synthetic polymer, such ascollagen, cellulosic membranes, polycarbonate, polytetrafluoroethylene,nylon membranes and nylon mesh. Other porous materials which can be usedas a cell-growth substrate are glass filters and ceramics. Preferably,the cell-growth substrate comprises a microporous membrane. Oneparticularly suitable support is Millicell-CM™ microporous insert(Millipore Corporation, Bedford, Mass.). In addition to membranes,filters can be used and are included within the scope of the invention.In a preferred embodiment, the cell-growth substrate is coated with acell-growth supporting material, such as collagen, laminin orfibronectin.

The surface of the porous cell-growth substrate must be favorable forcell growth. In particular, a cell-growth substrate will support growthbecause: its natural properties are sufficient; or it is treated toprovide cell growth properties; or it is coated with a cell-growthsupporting material, such as collagen. The surface of the substrate isthen activated to provide sites for attachment of growth factors.

In one embodiment, porous cell-growth substrate can be treated(chemically or non-chemically) to provide activated sites for attachinggrowth factors to the cell-growth substrate. The method by which acell-growth substrate is treated to provide activated sites foradherence of growth factors depends upon the substrate. A growth factoris any molecule which facilitates growth of cells of interest, such ascell-specific growth factors (for example, epidermal growth factor),hormones, cell culture medium, peptides, carbohydrates andglycoproteins.

In another embodiment, the cell-growth substrate is coated with acell-growth supporting material which is treated for attaching growthfactors thereto. Suitable cell-growth supporting materials areproteinaceous materials, such as collagen, gelatin, laminin andfibronectin. When the cell-growth substrate is coated with collagen, thecollage can be chemically treated by crosslinking it with gluteraldehydeor other crosslinking agents to provide activated sites for attachingthe growth factors thereto. Non-chemical methods, such as exposure toradiation, can be used to activate sites on the substrate.

A cell-growth substrate having activated attachment sites can then becontacted with cell culture medium comprising growth factor specific forcells of interest, under conditions whereby the growth factor isdispersed within the substrate. The cell culture medium can contain onetype of growth factor or it may contain a number of different types ofgrowth factors which are specific for cells of interest. The growthfactors can be dispersed throughout the substrate or they can bedispersed to areas of the substrate which will actually contact thecells, such as the surface of the cell-growth substrate. When thesubstrate is coated with crosslinked collagen, it is believed that thegrowth factors chemically attach themselves (e.g., by covalentattachment) to the activated sites located on the collagen. However,other means of attaching growth factors to the substrate are embraced bythis invention.

To insure that all activated sites on the substrate have beeninactivated or neutralized (i.e., all activated sites have been boundwith a growth factor), the cell culture medium is removed from thesubstrate and an additional amount of medium comprising growth factorsand non-specific protein is contacted with the substrate to saturateremaining activated sites. This step quenches or deactivates thesubstrate and insures a substantially uniform distribution of growthfactors within the substrate. Preferably, the non-specific protein isserum or albumin which can bind to remaining activated sites.

After the surface of the cell-growth substrate has been prepared, cellsof interest are seeded onto the substrate whereby the cells arecontacted with the growth factors to form a cell culture. The seededcell-growth substrate is then maintained under conditions suitable forcell growth to produce monolayer sheets of tissue. Further cell growthwill result in multilayered tissue which is differentiated. After thecells have grown for several days, the culture can be raised to theair/liquid interface where the cells can further differentiate to yieldtissue similar to its in vivo counterpart. Alternatively, the cells canbe maintained as a submerged culture. Tissue grown by the methods ofthis invention can be subsequently removed from the substrate andharvested.

In the preferred embodiment, tissue from keratinocyte cells can beproduced by providing a porous cell-growth substrate, such asmicroporous membrane, having a layer of collagen coated thereon. Thecollagen is treated (for example, by crosslinking it withgluteraldehyde) to provide activated sites for attaching growth factorsthereto. The treated collagen-coated substrate is then contacted withcell culture medium comprising epidermal growth factor and pituitaryextract which comprises growth factors therein, under conditions wherebythe growth factors are dispersed within the substrate and can bechemically attached thereto. Cell culture medium for proliferation ofnormal human epidermal keratinocytes (NHEK) containing epidermal growthfactor and bovine pituitary extract had been described by Boyce and HamU.S. Pat. No. 4,673,649, Jun. 16, 1987 and modified by Clonetics, Inc.(San Diego, Calif.) as Keratinocyte Growth Medium.

The cell culture medium is then removed from the substrate. Theresulting medium-free substrate is contacted with additional mediumwhich is modified with serum and calcium. The amount of medium added isthat which is sufficient to saturate remaining activated sites withgrowth factor and/or serum to thereby deactivate or quench thesubstrate. Once the substrate has been deactivated, keratinocyte cellsare seeded onto the substrate to form a cell culture. The cell seedingdensity can be from about 1×10² cells/cm² to about 1×10⁷ cells/cm².Preferably, keratinocyte cells are seeded at a seeding density of 1×10⁵cell/cm² to 8×10⁵ cells/cm². The culture is maintained under conditionssuitable for keratinocyte cell growth to thereby produce tissuecontaining keratinocyte cells. Tissue raised to the air/liquid interfacewill be stratified, terminally differentiated epidermis.

Toxicological effects of drugs, chemicals and cosmetics on mammaliancells is currently evaluated, in vivo. For example, toxicity ofcosmetics is tested using the Draize eye irritancy test on rabbits. As aresult of such tests, there is increasing public awareness and criticismof the use of animals for toxicity testing. In response to publicconcern, many countries have banned the use of animals for toxicology.Thus, alternative methods for screening toxic effects of drugs andchemicals must be developed.

Toxic effects of substance, such as drugs, chemicals and cosmetics, canbe evaluated using tissue produced by the methods of this invention. Asubstance to be evaluated is administered to tissue (from cells at anystage of growth and differentiation) grown on a cell-growth substrate,as previously described. Cellular response to the substance is observed.The response can be a change in cell morphology, growth and viability orit can be one or more chemical changes in the cell. The response is thenevaluated to determine toxic effects of the substance, such as bymeasuring the presence and amount of a biochemical or enzyme released inresponse to the substance. Dye release from the cells can also beindicative of toxicity. For example, a substance to be screened for itstoxic effects can be topically applied to stratified, terminallydifferentiated epidermis. Penetration of the skin layer by the substanceand release of a dye into the media or from the skin layer can then beassessed as indicative of toxicity. Alternatively, uptake of the dye bythe skin layer can be evaluated.

Additionally, since tissue produced by the methods of this inventionsufficiently, morphologically resemble in vivo tissue, it can be used inother physiological and clinical applications, such as to study cellulardevelopment into tissue, pharmacological mechanisms and transdermaltransport of drugs. The tissue can also be used as a tissue replacementfor wounds, burns and the like.

A kit for in vitro toxicology will comprise a porous cell-growthsubstrate having growth factors specific for growing cells of interestdispersed therein; tissue from cells of interest grown on the substrate;and reagents for determining the toxicity of a substance. Preferably,the substrate will be a crosslinked collagen gel coated on a microporouscell-growth substrate and the cells are keratinocyte cells which aregrown to produce terminally differentiated tissue. The tissue, however,can be readily available at various stages of cell growth from amonolayer to a stratified, multilayer tissue.

In another embodiment, the above kit can comprise a suspension of cellswhich can be seeded onto the cell-growth substrate and grown to therebyproduce tissue at any desired stage of cell growth. The cell-growthsubstrate minus the cells can be sold freeze-dried or frozen. The kitcan further comprise one or more reagents for determining toxicity of asubstance, such as buffers, detection compounds and indicator dyes(e.g., fluorescent compounds, radioactive labels, chemiluminescentcompounds). Preferably, the cell-growth substrate is a microporouspolymeric membrane and the cells are keratinocyte cells.

The invention will be further illustrated by the following examples.

EXAMPLE 1

Keratinocyte Growth on a Collagen-Coated Cell-Growth Substrate

Preparation of a Collagen-Coated Cell-Growth Substrate

Two parts by volume of collagen from rat tail Type I, about 3 mg/ml(Collaborative Research, Lexington, Mass.), was diluted with one part70% ethanol and mixed well by vortex. The resulting solution was sterileand could be stored refrigerated prior to use.

MilliCell-CM™ porous substrate inserts (Millipore Corporation, Bedford,Mass.) were placed in 100 mm petri dishes. 50 μl and 500 μl of thecollagen/ethanol mixture were added to petri dishes containing 12 mm and30 mm Millicell-CM™ inserts, respectively. Five or six drops ofconcentrated ammonium hydroxide were placed around the periphery of thepetri dishes. The petri dishes were then covered and incubated for 45minutes at room temperature to allow the collagen to gel.

The resulting collagen gel or gels were washed one time with 70%ethanol. The Millicell-CM™ inserts containing the collagen gel wereimmersed in 70% ethanol and incubated at room temperature for one hourto dehydrate the gel. The resulting gel was contracted on theMillicell-CM™ insert to form a dense gel. The gel was rinsed one timewith sterile water and then washed three times by aspirating the insertswith sterile phosphate buffered saline (PBS).

Crosslinking the Collagen

A 25% aqueous solution of gluteraldehyde was diluted 1:10 in PBS toyield a concentration of 2.5% gluteraldehyde in PBS. The resultingsolution was filtered to remove particulates. PBS was removed from theMillicell-CM™ inserts described in the previous section. The insertswere then immersed in sterile 2.5% gluteraldehyde solution and incubatedfor one hour to activate the surface of the collagen and crosslink it.The resulting crosslinked gel was washed three times with sterile PBS.

Attachment of Growth Factors to Gluteraldehyde Activated Collagen-CoatedSubstrate

PBS was removed from the crosslinked collagen gels as prepared in theprevious section. The crosslinked collagen gel was then immersed for 1.5hours in modified MCDB 153 nutrient medium (Keratinocyte Growth Medium(KGM), Clonetics, Inc., San Diego, Calif.; Ham U.S. Pat. No. 4,673,649,Boyce, S. and R Ham.). The medium contained Epidermal Growth Factor(EGF) and Bovine Pituitary Extract (BPE).

After 1.5 hours, the medium was removed and replaced with a modified KGMsolution. The KGM solution was modified by adding 10% Fetal Bovine Serum(FBS) and 1.5 mM calcium chloride from 200 mM stock solution of tissueculture grade calcium chloride in water. The collagen gel was thenimmersed in the modified KGM solution for one hour to quench anyremaining active sites on the crosslinked collagen gel.

Seeding the Cell-Growth Substrate

Normal human epidermal keratinocytes (NHEK) were acquired as secondarypassaged cell strains from Clonetics, Inc. The secondary cultures werefed daily with serum-free KGM and were used to seed crosslinked collagengels, prepared as described above, when the cultures were between 60-80%confluent. The KGM medium of the seed culture was replaced with freshKGM medium prior to seeding the collagen gel.

The keratinocytes were released from the culture flask with trypsin/EDTA(ethylenediaminetetraacetic acid) and prepared as a single cellsuspension in modified KGM (10% FBS, 1.5 mM calcium). The cells werethen seeded on the collagen gel substrate at a keratinocyte seedingdensity of 3-6×10⁵ cells/cm².

Growth of Keratinocytes on the Cell-Growth Substrate

The seeded collagen gel substrate was incubated at 37° C. The culturewas maintained as a submerged culture and was replenished daily withfresh solution of modified KGM (10% FBS, 1.5 mM calcium).

The keratinocyte cells attached to the collagen substrate within 24hours. The submerged culture of cells grew to a confluent monolayer andbegan to stratify between days 4-8. The cultures also exhibited asignificant electrical resistance between days 4-8, of about 150-200ohms cm², which indicated that the keratinocyte cell sheet was uninformand coherent. The cultures were raised to the air/liquid interfacebetween days 4-8 for further differentiation and keratinization of theculture.

FIGS. 1 and 2 are transmission electron micrographs of a keratinocytesheet raised to the air/liquid interface for 14 days. The micrographsshow a stratified, terminally differentiated sheet of keratinocytecells. (i.e., epidermis). At the air/liquid interface, the sheet hascornified envelopes which are characteristic of human epidermis, invivo. Cells which are several layers down from the air/liquid interfacecontain keratinohyalin granules (dense, black intracellular bodies).There are also numerous desmosomal junctions. In the basal portion ofthe sheet (FIG. 2) at the collagen gel interface, there is a smoothbasal membrane. Inside the basal membrane are numerous keratin filamentsand mitochondria. Directly beneath the basal membrane is a black linewhich ultrastructurally appears to be the formation of a basementmembrane.

FIG. 3 shows a hematoxylin and eosin stained histological cross-sectionof a uniform sheet of keratinocytes grown on the crosslinkedcollagen-coated cell-growth substrate (Millicell-CM™ microporousmembrane) as described above. Note that the layer of keratinocyte cellsand collagen are of uniform thickness. In a similar experiment, (resultsnot shown), keratinocyte cells were grown on an unmodified,uncrosslinked collagen-coated cell-growth substrate (Millicell-CM™microporous membrane). A histological cross-section of substrate showeda disorganized mass of keratinocyte cells. In addition, the collagenunder the cell mass appeared to be thinning, which may indicatecollagenase degradation.

EXAMPLE 2

In Vitro Toxicological Study

Cell Culture

Madin-Darby Canine Kidney cells (MDCK, ATCC No. 34) were cultured inDulbecco's Modified Eagle's Medium (DMEM) with 10% FBS. Normal humanepidermal keratinocytes (NHEK) (Clonetics, Inc., San Diego, Calif.) werecultured in KGM with 10% FBS and 1.7 mM calcium. Both cell types weregrown on collagen-coated microporous Millicell-CM™ culture plate insertsand Biopore™ membranes (Millipore Corporation, Bedford, Mass.). Thecollagen-coated microporous membranes were prepared as described inExample 1. Both cell types were seeded at 5×10⁵ cells/cm².

MDCK and NHEK cells grown on the collagen-coated membranes exhibited adifferentiated in vivo-like ultrastructure characterized by cuboidalmorphology, basal nucleus, desmosomes and tight junctions (in MDCKcells) and stratification (in NHEK cells). MDCK cells were used forcytotoxicity studies at confluence, 4 days after seeding. NHEK cellswere used for similar studies at 8 days after seeding.

Cell Staining

Rhodamine 123 (a mitochondrial stain) was diluted in Earle's BalancedSalt Solution (EBSS, 10 μg/ml). Both cell types were stained for 1 hourwith the rhodamine solution at room temperature. Cells were washed priorto cytotoxicity testing.

BCECF-AM (an analog of 6-carboxyfluorescein diacetate and anintracellularly fluorescent stain of viable cells; Molecular Probes,Inc., Eugene, Oreg.) was diluted in serum-free, phenol red-freeDulbecco's Modified Eagle Medium (DMEM) (40 μg/ml) and used to stainboth cell types for 35 minutes at room temperature. Cells were rinsedprior to cytotoxicity testing.

Cytotoxicity Testing and Detection

Dilutions (0-100 μM) of either mercuric chloride or cadmium chloride inEBSS were applied only to the apical membrane surface. In similarexperiments, dilutions of either 10% Sodium dodecyl sulfate (SDS) orTween 20 (Sigma Chemical, St. Louis, Mo.) in DHEM were applied only tothe apical membrane surface.

Dye release as an indicator of cytotoxicity was measured using aFluoroskan II Spectrofluorimeter (Flow Labs, Inc., McLean Va.).Supernatant containing released fluorescent dye was read directly usingan excitation wavelength of 485 nm and an emission wavelength of 538 nm.

Cytotoxicity Results

The dose response efflux of the fluorescent probes, such as rhodamine123 and carboxyfluorescein diacetate analogs, was measured byquantitifying fluorescene released to the apical compartment. The effluxwas indicative of the degree of mitochondrial toxicity and cell membranedamage in response to heavy metals (i.e., mercuric chloride or cadmiumchloride) and detergents (i.e., SDS or Tween).

With rhodamine 123 probes, mitochondrial toxicity preceded disruption ofplasma membrane integrity. Using fluorescent probes of BCECF-AM, acuteapical plasma membrane damage was observed by release of fluorescence tothe apical compartment. Extensive membrane damage is indicated byrelease of fluorescence to both the apical and basal compartments.

For 10% SDS, assay sensitivity extended to dilutions of 1 in 10,000.

FIG. 4 shows the dose response curve for the apical and basal membraneof keratinocyte cells exposed to various concentrations of SDS.Keratinocyte sheets were grown on collagen-coated microporous membranesand were stained with fluorescent dye C-1354 an analog ofcarboxyfluorescein (Molecular Probes, Inc., Eugene, Oreg.). Damage tothe cell was assessed by measuring the fluorescene in both the apicaland basal compartments.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

I claim:
 1. A kit for evaluating toxic effects of a substance on tissue,in vitro, comprising:a. a polymeric microporous cell growth substratethat is coated with a cell growth supporting material which has beentreated for attaching growth factors thereto, the coated substrate beingsuitable for growing keratinocyte cells thereon and having growth factorspecific for growing the cells dispersed therein and attached to thesupport material; b. tissue from keratinocyte cells grown on the coatedsubstrate of (a), in the absence of a second cell type, wherein thetissue is grown as a confluent monolayer of tissue or uniformlydifferentiated multilayer tissue in a submerged culture or at the airliquid interface; and c. one or more reagents for determining toxicityof the substance.
 2. The kit of claim 1, wherein the microporoussubstrate is a microporous polymeric membrane that is coated withcollagen comprising activated sites for attaching the growth factorthereto.
 3. A kit for evaluating toxic effects of a substance on tissue,in vitro, comprisinga. a collagen-coated polymeric microporous cellgrowth substrate that has been treated for attaching growth factorsthereto, the coated substrate being suitable for growing keratinocytecells thereon, in the absence of a second cell type, and having growthfactor specific for growing the cells dispersed therein and attached tothe collagen; and b. one or more reagents for determining toxicity ofthe substance.
 4. A method for determining toxic effects of a substanceon tissue, in vitro, comprising the steps of:a. providing a tissue fromkeratinocyte cells which is grown, in the absence of a second cell type,on a microporous cell growth substrate that is coated with a cell growthsupporting material which has been treated for attaching growth factorsthereto, the coated substrate being suitable for growing keratinocytecells thereon and having growth factor dispersed within the supportmaterial and attached thereto, the growth factor being specific forkeratinocyte cell growth, wherein the tissue is grown as a confluentmonolayer of tissue or uniformly differentiated multilayer tissue in asubmerged culture or at the air liquid interface; b. exposing the tissueto a substance to be tested; and c. evaluating cellular response to thesubstance to determine the toxicity thereof.
 5. The method of claim 4,wherein the substrate is a microporous polymeric membrane which iscoated with collagen comprising activated sites for attaching the growthfactor thereto.
 6. A method for determining toxic effects of a substanceon tissue, in vitro, comprising the steps of:a. providing a collagencoated polymeric microporous cell growth substrate that is coated with acell growth supporting material which has been treated for attachinggrowth factors thereto; the coated substrate being suitable for growingkeratinocyte cells and having growth factor dispersed within thecollagen and attached thereto, the growth factor being specific forgrowing the cells on the coated substrate; b. subsequently seedingkeratinocyte cells onto the coated microporous substrate and maintainingthe seeded microporous cell growth substrate under conditions suitablefor cell growth, in the absence of a second cell type, to therebyproduce a confluent monolayer of tissue or uniformly differentiatedmultilayer tissue; and c. exposing the tissue to the substance andevaluating cellular response to the substance to determine the toxiceffects thereof.